Peptides and therapeutic agent for autoimmune diseases containing the same

ABSTRACT

The present invention relates to a peptide having the following amino acid sequence: 
     Ala-Xaa1-Leu-Xaa2-Phe-Xaa3-Xaa4-Xaa5-(Xaa6)n 
     (wherein Xaa1 and Xaa4 each independently represents an amino acid residue which may have an alkyl or heteroalkyl side chain which may be substituted by a hydroxy, amino or guanidyl group; 
     Xaa2 and Xaa6 each independently represents an amino acid residue which may have an alkyl or heteroalkyl side chain which may be substituted by a hydroxyl group; 
     Xaa3 and Xaa5 each independently represents an amino acid residue which may have a hydrophobic side chain; and 
     n stands for 1 or 0), or derivatives thereof; and a modification thereof. 
     The peptide or derivatives thereof according to the present invention is useful as a pharmaceutical composition for the prevention and treatment of autoimmune diseases, rejection reaction attendant on the organ transplantation, inflammation or the like.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to peptides or derivatives thereof. Thepeptides or derivatives thereof according to the present invention areuseful for the antigen non-specific suppressive treatment of abnormallyaugmented immunoreaction in autoimmune diseases. Having ananti-inflammatory effect, it is also useful for the treatment of theinflammation.

Accordingly, the present invention relates to the fields concerned withpeptides or derivatives thereof and also with pharmaceuticals containingthe same.

1. Background of the Invention

Autoimmune diseases are induced by the continuous production of anantibody or lymphocyte which reacts with a component of the own tissue.In the autoimmune diseases, described specifically, the break-down ofimmunologic tolerance heightens immune response to own organiccomponents, which causes the reaction between an autoantibody orautoreactive T cell so produced and an autoantigen or cell correspondingthereto, thereby causing cellular dysfunction or tissue damages. Atpresent, 50 or more types of autoimmune diseases are known and accordingto the spreading degree of a lesion over the organs, they can beclassified into organ specific autoimmune diseases and organ nonspecificautoimmune diseases.

Examples of the former diseases include insulin-dependent diabetesmellitus in which a lesion is caused by the selective destruction of Bcells in pancreatic island of Langerhans, Basedow disease and Hashimotodisease in which thyroid dysfunction is caused by the antibody against athyroid stimulating hormone receptor, myasthenia gravis which has musclecontraction lowered by an antibody against an acetylcholine receptor ofthe striated muscle, and autoimmune hemolytic anemia in which hemolysisis caused by the antibody against the erythrocyte.

Examples of the latter diseases include chronic rheumatoid arthritis inwhich generalized disorders in the osseous or cartilaginous tissue areconsidered to occur, triggered by the aggregation of IgG and anti-IgGantibodies (rheumatoid factors), systemic lupus erythematosus in which adisturbing reaction is caused by the deposition of antibodies againstDNA or nuclear components to the kidney, joint or skin, SjUgren syndromein which dysfunction occurs owing to the lymphocytic infiltration intothe salivary gland or lacrimal gland and systemic organolesion such asinterstitial nephritis occurs concurrently at certain frequency, andmultiple sclerosis in which disseminated demyelination nidi and gliosisappear in the substantia alba of the central nervous system and theycause systemic motor paralysis, ophthalmopathy, paresthesia or the like.

2. Prior Art

For the treatment of autoimmune diseases, it is the common practice toadminister an immunosuppressant typified by a gluco-steroid preparation,cyclosporin A or FK 506. The treatment using such a preparation is,however, accompanied by the drawback such as serious side effects, forexample, infectious diseases, nephrotoxicity of the drug itself orcarcinogenesis, which result from wide spectrum of immunosuppression[Sadao Kashiwazaki, Sogo Rinsho, 43 (9), 1725-1729 (1994)].

In recent years, there have been some attempts to treat autoimmunediseases without using such an immunosuppressant with wide spectrum.

Upon reaction with an antigen, B cells recognize the antigen itself,while T cells recognize the complex of an MHC molecule on the surface ofthe antigen presenting cell and an antigen peptide fitted in the grooveof the MHC molecule. The MHC molecule differs with individuals and humanT cells having MHC congenial to a certain antigen shows good response tothis antigen. This is one of the reasons why some human beings arelikely to be reactive to a certain antigen. On the other hand, T-cellantigen receptors (T cell receptors: TcR) can be classified into severalfamilies. There is a substance which activates T cells, binding with oneor some of the TcR families and the substance is called a superantigen.The superantigen activates a larger number of T cells compared withthose in the case of the ordinary immunoreaction so that it happens tocause a large reaction, leading to a disease. For the activation of Tcells, binding of a partner molecule (ligand) to the adhesion moleculeon the surface is necessary in addition to the recognition of theantigen. The reaction of T cells can, therefore, be inhibited byblockading the adhesion molecule, or the reaction can be amplified bythe enhanced expression of the ligand of the partner cell.

It is reported that among T cell groups, there exist T cells whichsuppress the immunoreaction which are so-called suppressor T cells [Tadaand Takemori, J. Exp. Med., 140, 239 (1974)]. It is elucidated thatthese T cells produce soluble immunosuppressive factors for suppressingantigen specific antibody production [Tada, et al., J. Immul., 111, 952(1973)]. The relationship between the soluble immunosuppressive factorsand a TcR alfa chain is reported [Dorf, et al., J. Immunol., 145,2809-2819 (1990)]. Experimental allergic encephalomyelitis (EAE) whichis a model of multiple sclerosis is induced by the administration ofbasic myelin protein. In the EAE, presence of a T cell antigen receptor(disease specific TcR) which is expressed specifically on the pathogeniclymphocyte is known. The treatment method by using an antibody against adisease-specific TcR or TcR peptide vaccine has, therefore, beendeveloped [Howell, et al., Science, 251, 430-432 (1991); Vandenbrk, etal., Nature, 341, 541544(1989)]. Such a treatment method of autoimmunediseases is considered to be associated with less serious side effectscompared with the method employing an immunosuppressant such asgluco-steroid preparation or cyclosporin A. The above method can only beapplied to the case where the T cell or antigen which has caused adisease is circumscribed within a markedly narrow range. It isconsidered to be difficult to apply the above method to autoimmunediseases, such as chronic rheumatoid arthritis, of which antigen is notyet determined and in which a plural number of pathogenic lymphocytesexist. This method requires the establishment of individual suppressor Tcell specific to each antigen and analysis of its TcR. Under the presentstate where the suppressor T cells derived from human being cannot beestablished easily, it is extremely difficult to develop the above TcRas an immunosuppressive agent.

On the other hand, reported is a case where immunologic tolerance isinduced by using a peptide having an antigen-derived amino acid sequence[Greenstein, et al., Proc. Natl. Acad. Sci. USA, 9, 7608-7612 (1993)].As a method of using an immunosuppressant composed of a TcR peptidefragment, disclosed is a method in which TcR beta-chain derivedpolypeptide (composed of 13 amino acid residues at the minimum) is usedfor the treatment of autoimmune diseases, particularly, chronicrheumatoid arthritis (Japanese Language Laid-Open Publication No. HEI6-511241); and a method in which a TcR peptide fragment of pathogenic Tcells which cause multiple sclerosis is administered to suppress theonset of EAE (WO90/11294). Mohapatra et al. have proved that a syntheticpeptide composed of 15 amino acid residues containing CDR3(complementarity determining region), which is a specific antigendetermining site of TcR derived from suppressor T-cell, has animmunosuppressive effect [J. Immunol., 151, 688-698 (1993)].

The above-described peptide fragments are no better than animmunosuppressive agent characterized by having antigen specificity sothat a problem that it cannot be used for the treatment of all theautoimmune diseases has remained unsolved.

SUMMARY OF THE INVENTION

An object of the present invention is to overcome the above describeddisadvantages or defects of the conventional methods, thereby providinga therapeutic agent for autoimmune diseases, which has antigennonspecific immunosuppressive effects and can therefore be used for thetreatment of autoimmune diseases of which antigens are not yet beendetermined.

Another object of the present invention is to provide a therapeuticagent for autoimmune diseases, which can be used for the treatment ofautoimmune diseases accompanied with inflammation.

A further object of the present invention is to provide a peptide whichis characterized by having a low molecular weight compared with thatemployed in the conventional method so that it can be prepared at a lowcost and has markedly reduced side effects such as occurrence ofantigenecity due to frequent administration.

As a result of an extensive investigation on the mutual relationshipbetween the amino acid sequence of a protein or peptide fragmentoriginated from a protein such as CDR3 of TcR derived from suppressor Tcells and a therapeutic agent for autoimmune diseases, the presentinventors have found that there exist some peptides having suppressiveactivity against antigen nonspecific IgG production. Finding that apeptide composed of 9 amino acid residues, which is shown as Sequence IDNo. 1 in the Sequence Listing, suppresses not only the anti-ovalbumin(OVA) antibody production but also anti-keyhole-lympet-hemocyanin (KLH)antibody production in each in vivo experiment, the present inventorshave completed the present invention. Incidentally, OVA and KLH aretypical antigens used as immunogens (Immunology Dictionary, 1993, TokyoKagaku Dojin, pp101 and 130).

In general, TcR derived from suppressor T cells corresponds with one byone specifically to various antigens. The peptide of the presentinvention, on the other hand, suppresses antibody production againstdifferent antigens such as OVA and KLH and, therefore, has antigennonspecific immunosuppressive activity.

The present inventors have also found that the peptide composed of 9amino acid residues, which is shown as Sequence ID No. 1 in the SequenceListing, suppresses the onset of experimental allergic encephalomyelitis(EAE), a model of multiple sclerosis, which is one of autoimmunediseases for which T cells are responsible. In addition, it issurprising that they have found the peptide composed of 9 amino acidresidues, which is indicated by Sequence ID No. 1 in the SequenceListing, suppresses the edema in a carrageenin-induced paw edema modeland, therefore, has an anti-inflammatory effect. The peptide of thepresent invention has a property of immunosuppression by suppressing Tcell activities as well as anti-inflammation.

As a result of extensive efforts to develop a low-molecular weightpeptide which has less antigenicity and can be easily synthesized, thepresent inventors have found that the peptide composed of 8 amino acidresidues, which is shown as Sequence ID No. 2 in the Sequence Listing,suppresses not only the anti-OVA antibody production but also anti-KLHproduction, in each in vivo experiment. The present inventors have alsofound by the alanine substitution method [Geysen H. M., et al., J.Immunol. Methods, 102, 259-274 (1987)] that there exist, in somepeptides having a certain consensus amino acid sequence, peptides havingan antigen nonspecific IgG production suppressive activity. Finding thatsuch peptides having a general amino acid sequence suppresses not onlythe anti-ovalbumin (OVA) antibody production but alsoanti-keyhole-lympet-hemocyanin (KLH) antibody production in each in vivoexperiment, the present inventors have completed the present invention.

Accordingly, the present invention relates to a peptide having an aminoacid sequence represented by the following formula:

Ala-Xaa1-Leu-Xaa2-Phe-Xaa3-Xaa4-Xaa5-(Xaa6)n

(wherein Xaa1 and Xaa4 each independently represents an amino acidresidue which may have an alkyl or heteroalkyl side chain which may besubstituted by a hydroxy, amino or guanidyl group;

Xaa2 and Xaa6 each independently represents an amino acid residue whichmay have an alkyl or heteroalkyl side chain which may be substituted bya hydroxyl group;

Xaa3 and Xaa5 each independently represents an amino acid residue whichmay have a hydrophobic side chain; and

n stands for 1 or 0),

or derivatives thereof; a pharmaceutical composition containing thesame; and a method for the treatment of diseases using thereby.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart of the chromatogram of Peptide (I). Peptide (I)obtained according to the present invention had a purity of 100%.

FIG. 2 is a chart of the chromatogram of Peptide (II). Peptide (II)obtained according to the present invention had a purity of 99.6%.

FIG. 3 is a chart of the chromatogram of Peptide (III). Peptide (III)obtained according to the present invention had a purity of 98.8%.

FIG. 4 is a chart of the chromatogram of Peptide (IV). Peptide (IV)obtained according to the present invention had a purity of 97.2%.

FIG. 5 is a chart of the chromatogram of Peptide (V). Peptide (V)obtained according to the present invention had a purity of 98.5%.

FIG. 6 is a chart of the chromatogram of Peptide (VI). Peptide (VI)obtained according to the present invention had a purity of 98.4%.

FIG. 7 is a chart of a chromatogram of Peptide (VII). Peptide (VII)obtained according to the present invention had a purity of 97.9%.

FIG. 8 is a chart of the chromatogram of Peptide (VIII). Peptide (VIII)obtained according to the present invention had a purity of 98.1%.

FIG. 9 is a chart of the chromatogram of Peptide (IX). Peptide (IX)obtained according to the present invention had a purity of 97.5%.

FIG. 10 is a chart of the chromatogram of Peptide (X). Peptide (X)obtained according to the present invention had a purity of 98.2%.

FIG. 11 is a chart of the chromatogram of Peptide (XI). Peptide (XI)obtained according to the present invention had a purity of 97.6%.

FIG. 12 is a chart of the chromatogram of Peptide (XII). Peptide (XII)obtained according to the present invention had a purity of 97.5%.

FIG. 13 is a chart of the chromatogram of Peptide (XIII). Peptide (XIII)obtained according to the present invention had a purity of 98.2%.

FIG. 14 is a chart of the chromatogram of Peptide (XIV). Peptide (XIV)obtained according to the present invention had a purity of 98.6%.

FIG. 15 is a chart of the chromatogram of Peptide (XV). Peptide (XV)obtained according to the present invention had a purity of 100%.

DISCLOSURE OF THE INVENTION

The present invention relates to a peptide having an amino acid sequencerepresented by the following formula:

Ala-Xaa1-Leu-Xaa2-Phe-Xaa3-Xaa4-Xaa5-(Xaa6)n

(wherein Xaa1 and Xaa4 each independently represents an amino acidresidue which may have an alkyl or heteroalkyl side chain which may besubstituted by a hydroxy, amino or guanidyl group;

Xaa2 and Xaa6 each independently represents an amino acid residue whichmay have an alkyl or heteroalkyl side chain which may be substituted bya hydroxyl group;

Xaa3 and Xaa5 each independently represents an amino acid residue whichmay have a hydrophobic side chain; and

n stands for 1 or 0, with the proviso that the case where Xaa2represents glycine is excluded),

or a derivative thereof.

Xaa1 and Xaa4 each independently represents an amino acid residue whichmay have an alkyl or heteroalkyl side chain which may be substituted bya hydroxy, amino or guanidyl group. Examples may include Lys, Arg, Hisand Ala.

Xaa2 and Xaa6 each independently represents an amino acid residue whichmay have an alkyl or heteroalkyl side chain which may be substituted bya hydroxyl group. Examples may include Thr and Ala.

Xaa3 and Xaa5 each independently represents an amino acid residue whichmay have a hydrophobic side chain. Examples may include Gly and Ala.

The term "alkyl" as used herein means a group which embraces primary,secondary and tertiary saturated C1-15, preferably C1-10 alkyl groupswhich may be linear or branched. The term "heteroalkyl" as used hereinmeans a group in which at least one of the carbon atoms of theabove-described C1-15, preferably C1-10 "alkyl" group has beensubstituted with a like number of hetero atoms such as sulfur (S) oroxygen (O) [ordinarily, in the form of a (thio)ester or (thio)ether]. nstands for 1 or 0.

In the peptide derivative of the present invention, all or part of theamino acids may be in the D form, a functional group having an activehydrogen may be substituted with an appropriate protective group or thecarboxyl group at the C-terminal may be in the form of a carboxyderivative such as amide or ester. No particular limitation is imposedon the protective group suitable for the substitution with the amino orcarboxyl group in the peptide, however, an acetyl group (Ac) ort-butoxycarbonyl group (tBoc) is preferred. As a carboxy derivative atthe C terminal, an amide is preferred.

The conversion into a peptide derivative can bring about an improvementin the stability.

Described specifically, the present invention pertains to a peptidehaving the following amino acid sequence;

Ala-Xaa1-Leu-Xaa2-Phe-Xaa3-Xaa4-Xaa5-(Xaa6)n

(wherein Xaa1 and Xaa4 each independently represents any one of Lys,Art, His and Ala;

Xaa2 and Xaa6 each independently represents Thr or Ala;

Xaa3 and Xaa5 each independently represents Gly or Ala; and n stands for1 or 0), or derivative thereof.

More specifically, the present invention relates to a peptide having anamino acid sequence selected from the group consisting of the followingamino acid sequences:

Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr (Seq. ID No. 1),

Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 2),

Ala-Ala-Leu-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 3),

Ala-Lys-Leu-Ala-Phe-Gly-Lys-Gly (Seq. ID No. 4),

Ala-Lys-Leu-Thr-Phe-Ala-Lys-Gly (Seq. ID No. 5),

Ala-Lys-Leu-Thr-Phe-Gly-Ala-Gly (Seq. ID No. 6), and

Ala-Lys-Leu-Thr-Phe-Gly-Lys-Ala (Seq. ID No. 7).

Still more specifically, the present invention relates to a peptide orderivative thereof selected from the group consisting of the peptidesand derivatives thereof represented by the following formulas (I) to(XII):

    Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr (Seq. ID No. 1)        (I)

    DAla-DLys-DLeu-DThr-DPhe-Gly-DLys-Gly-DThr                 (II)

    Ac-Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr                     (III)

    tBoc-Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr                   (IV)

    Ac-DAla-DLys-DLeu-DThr-DPhe-Gly-DLys-Gly-DThr              (V)

    Ac-DAla-DLys-DLeu-DThr-DPhe-Gly-DLys-Gly-DThr-NH2          (VI)

    Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 2)            (VII)

    Ala-Ala-Leu-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 3)            (VIII)

    Ala-Lys-Leu-Ala-Phe-Gly-Lys-Gly (Seq. ID No. 4)            (IX)

    Ala-Lys-Leu-Thr-Phe-Ala-Lys-Gly (Seq. ID No. 5)            (X)

    Ala-Lys-Leu-Thr-Phe-Gly-Ala-Gly (Seq. ID No. 6)            (XI)

    Ala-Lys-Leu-Thr-Phe-Gly-Lys-Ala (Seq. ID No. 7)            (XII)

(wherein D represents a D-form, Ac represents an acetyl group and tBocrepresents a t-butoxycarbonyl group),

or a derivative thereof.

No particular limitation is imposed on the preparation process of thepeptide having an amino acid sequence represented by the followingformula:

Ala-Xaa1-Leu-Xaa2-Phe-Xaa3-Xaa4-Xaa5-(Xaa6)n

(wherein Xaa1 and Xaa4 each independently represents an amino acidresidue which may have an alkyl or heteroalkyl side chain which may besubstituted by a hydroxy, amino or guanidyl group;

Xaa2 and Xaa6 each independently represents an amino acid residue whichmay have an alkyl or heteroalkyl side chain which may be substituted bya hydroxyl group;

Xaa3 and Xaa5 each independently represents an amino acid residue whichmay have a hydrophobic side chain; and

n stands for 1 or 0),

or a derivative thereof. For example, it is possible to synthesize apeptide by using a peptide synthesizer in accordance with thesolid-phase peptide synthesis (Fmoc) in accordance with an ordinarymanner, followed by purification by a reverse-phase HPLC column.Peptides (II) to (XII) can also be synthesized by the peptidesynthesizer and they can be chemically converted as needed.

The peptide of the present invention is preferred to have solublephysicochemical properties. Because the peptide of the present inventionhas a low molecular weight, side effects such as antigenicity occurrencecan be significantly reduced even after frequent administration andbesides, its toxicity is very low. The present invention also pertainsto a pharmaceutical composition containing a peptide having an aminoacid sequence represented by the following formula:

Ala-Xaa1-Leu-Xaa2-Phe-Xaa3-Xaa4-Xaa5-(Xaa6)n

(wherein Xaa1 and Xaa4 each independently represents an amino acidresidue which may have an alkyl or heteroalkyl side chain which may besubstituted by a hydroxy, amino or guanidyl group;

Xaa2 and Xaa6 each independently represents an amino acid residue whichmay have an alkyl or heteroalkyl side chain which may be substituted bya hydroxyl group;

Xaa3 and Xaa5 each independently represents an amino acid residue whichmay have a hydrophobic side chain; and

n stands for 1 or 0),

or a derivative thereof; or a pharmaceutical composition containing saidpeptide or derivative thereof and a pharmaceutically acceptable carrier.

Described specifically, the present invention pertains to a peptidehaving an amino acid sequence represented by the following formula:

Ala-Xaa 1-Leu-Xaa2-Phe-Xaa3-Xaa4-Xaa5-(Xaa6)n

(wherein Xaa1 and Xaa4 each independently represents any one of Lys,Arg, His and Ala;

Xaa2 and Xaa6 each independently represents Thr or Ala;

Xaa3 and Xaa5 each independently represents Gly or Ala; and

n stands for 1 or 0),

or derivative thereof.

More specifically, the present invention pertains to a pharmaceuticalcomposition containing a peptide having an amino acid sequence selectedfrom the group consisting of the following amino acid sequences:

Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr (Seq. ID No. 1),

Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 2),

Ala-Ala-Leu-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 3),

Ala-Lys-Leu-Ala-Phe-Gly-Lys-Gly (Seq. ID No. 4),

Ala-Lys-Leu-Thr-Phe-Ala-Lys-Gly (Seq. ID No. 5),

Ala-Lys-Leu-Thr-Phe-Gly-Ala-Gly (Seq. ID No. 6) and

Ala-Lys-Leu-Thr-Phe-Gly-Lys-Ala (Seq. ID No. 7),

or derivatives thereof; or a pharmaceutical composition containing saidpeptide or derivatives thereof and a pharmaceutically acceptablecarrier.

Still more specifically, the present invention also pertains to apharmaceutical composition containing a peptide or derivatives thereofselected from the group consisting of peptides and derivatives thereofrepresented by the following formulas (I) to (XII):

    Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr (Seq. ID No. 1)        (I)

    DAla-DLys-DLeu-DThr-DPhe-Gly-DLys-Gly-DThr                 (II)

    Ac-Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr                     (III)

    tBoc-Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr                   (IV)

    Ac-DAla-DLys-DLeu-DThr-DPhe-Gly-DLys-Gly-DThr              (V)

    Ac-DAla-DLys-DLeu-DThr-DPhe-Gly-DLys-Gly-DThr-NH2          (VI)

    Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 2)            (VII)

    Ala-Ala-Leu-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 3)            (VIII)

    Ala-Lys-Leu-Ala-Phe-Gly-Lys-Gly (Seq. ID No. 4)            (IX)

    Ala-Lys-Leu-Thr-Phe-Ala-Lys-Gly (Seq. ID No. 5)            (X)

    Ala-Lys-Leu-Thr-Phe-Gly-Ala-Gly (Seq. ID No. 6)            (XI)

    Ala-Lys-Leu-Thr-Phe-Gly-Lys-Ala (Seq. ID No. 7)            (XII)

(wherein D represents a D-form, Ac represents an acetyl group and tBocrepresents a t-butoxycarbonyl group);

or a pharmaceutical composition containing said peptide or derivativesthereof and a pharmaceutically acceptable carrier.

In the peptide derivatives used for the pharmaceutical composition ofthe present invention, all or part of the amino acids may be in the Dform, a functional group having an active hydrogen may be substitutedwith an appropriate protective group or the carboxyl group at theC-terminal may be in the form of a carboxy derivative such as amide orester. No particular limitation is imposed on the protective groupsuitable for the substitution with the amino or carboxyl group in thepeptide, however, an acetyl group (Ac) or t-butoxycarbonyl group (tBoc)is preferred. As a carboxy derivative at the C terminal, an amide ispreferred. The conversion into a peptide derivative can bring about animprovement in the stability.

The above-described pharmaceutical composition of the present inventionis useful particularly for the treatment of autoimmune diseases.Accordingly, the present invention provides a pharmaceutical compositionfor the treatment of autoimmune diseases.

The pharmaceutical composition of the present invention for thetreatment of autoimmune diseases is characterized by its antigennonspecificity. The peptide or derivatives thereof according to thepresent invention suppress the activity of T-cells so that the presentinvention also relates to a pharmaceutical composition for the treatmentof one or more than one diseases selected from the group consisting ofmultiple sclerosis, chronic rheumatoid arthritis, systemic lupuserythematosus, SjUgren syndrome, Basedow disease, Hashimoto disease andautoimmune hemolytic anemia.

The above-described pharmaceutical composition of the present inventionis also useful for the prevention and treatment of rejection episodeattendant to the organ transplantation. Accordingly, the presentinvention provides a pharmaceutical composition for the prevention andtreatment of rejection episode attendant to the organ transplantation.

Moreover, the above-described pharmaceutical composition of the presentinvention has an anti-inflammatory effect and is useful as ananti-inflammatory agent. Accordingly, the present invention provides apharmaceutical composition for the treatment of inflammation.

The present invention also relates to a method of treatment forautoimmune diseases, which comprises administering a pharmaceuticallyeffective amount of the above-described peptide or derivatives thereof.The present invention also relates to a method of treatment for one ormore than one diseases selected from the group consisting of multiplesclerosis, chronic rheumatoid arthritis, systemic lupus erythematosus,SjUgren syndrome, Basedow disease, Hashimoto disease and autoimmunehemolytic anemia, which comprises administering a pharmaceuticallyeffective amount of the above-described peptide or derivatives thereof.The present invention also pertains to a preventive and curative methodof rejection episode attendant to the organ transplantation, whichcomprises administering a pharmaceutically effective amount of theabove-described peptide or derivatives thereof.

The present invention also pertains to a method of treatment forinflammation, which comprises administering a pharmaceutically effectiveamount of the above-described peptide or derivatives thereof.

The present invention also relates to the use of the above-describedpeptide or derivatives thereof for the preparation of a pharmaceuticalcomposition for the treatment of autoimmune diseases. The presentinvention also relates to the use of the above-described peptide orderivative thereof for the preparation of a pharmaceutical compositionfor the treatment of one or more than one diseases selected from thegroup consisting of multiple sclerosis, chronic rheumatoid arthritis,systemic lupus erythematosus, SjUgren syndrome, Basedow disease,Hashimoto disease and autoimmune hemolytic anemia.

The present invention also relates to the use of the above-describedpeptide or derivatives thereof for the preparation of a pharmaceuticalcomposition for the prevention and treatment of rejection episodeattendant to the organ transplantation. The present invention alsorelates to the use of the above-described peptide or derivatives thereoffor the preparation of a pharmaceutical composition for the treatment ofinflammation.

Although the clinical dosage of the peptide of the present inventionvaries depending on the administration method, age, weight or conditionsof each patient, or the like, it typically falls within a range of 0.05to 500 mg, preferably 0.1 to 100 mg per day and adult.

As an administration method, intravenous administration can be employed.In addition to the ordinary intravenous injection, transfusion is alsopossible.

As an injection preparation, the peptide of the present invention or thederivatives thereof can be formulated, for example, into a powderypreparation for injection. In this case, the injection preparation canbe obtained by adding one or more than one suitable water-solubleexcipients selected from mannitol, sucrose, lactose, maltose, glucose,fructose, or the like added to the powdery preparation, dissolving theresulting mixture in water, pouring portions of the resulting solutionin vials or ampoules, lyophilizing and then hermetically sealing. It isalso possible to administer in systemic the powdery preparation throughthe nose or lungs as a fine-particulate aerosol preparation. Inaddition, the powdery preparation can be administered orally after addedwith a suitable excipient or the like.

In the present invention, the suppressive effects of anti-OVA antibodyproduction and anti-KLH antibody production in mice were studied by theELISA method. Compared with the non-administered group, the peptide ofthe present invention exhibited significantly high suppressive activityagainst anti-OVA antibody production. Even in comparison with a peptidefragment composed of 15 amino acid residues originated from CDR3, thepeptide of the present invention showed markedly high antibodyproduction suppressive activity. Compared with the non-administeredgroup, the peptide of the present invention exhibited markedly highsuppressive activity against anti-KLH antibody production.

In addition, the suppressive effects against the onset of experimentalallergic encephalomyelitis (EAE) in mice was examined. Compared with thenon-administered group, the peptide of the present invention showedsignificant suppressive effect against the onset of EAE. Moreover, thesuppressive effect of the peptide of the present invention on the ratcarrageenin-induced paw edema model was examined, resulting inconsiderably high edema suppressive effect compared with thenon-administered group.

It has not so far been reported that a low molecular weight peptidewhich is derived from a TcR alfa chain J region and can be artificiallysynthesized has an antigen nonspecific immunosuppressive effect. Thepeptide or derivatives of the present invention are useful as atherapeutic agent of autoimmune diseases, particularly, a pharmaceuticalcomposition for the treatment of one or more than one diseases selectedfrom the group consisting of multiple sclerosis, chronic rheumatoidarthritis, systemic lupus erythematosus, Sjugren syndrome, Basedowdisease, Hashimoto disease and autoimmune hemolytic anemia. It has alsoan anti-inflammatory effect so that it is useful as a therapeutic agentfor, among autoimmune diseases, those accompanied with inflammation suchas chronic rheumatoid arthritis. Moreover, the peptide of the presentinvention has antibody production suppressive effects so that it isuseful as a preventive or therapeutic agent for immediate type allergyin which IgE is concerned, such as pollinosis, atopic dermatitis,asthma, anaphylactic shock or hay fever, or drug allergy.

The present invention will hereinafter be described in detail byexamples but it should however be borne in mind the present invention isnot limited to or by the following examples.

Example 1 Synthesis of Peptides

Synthesis of Peptide (I)

Peptide (I) was synthesized by the solid-phase method (Fmoc) by using apeptide synthesizer ("Model 430 A", Applied Biosystems Inc.).

An amino acid having an N-terminal protected with a9-fluorenylmethyloxycarbonyl (Fmoc) group was coupled on ap-hydroxymethylphenoxymethylpolystyrene (HMP) resin by the method whichis described below.

First, the amino acid on the resin were deprotected at room temperaturefor 30 minutes by using 20% piperidine/N-methylpyrrolidone (NMP),followed by washing twice with NMP and then with 50% dichloromethane(DCM)/methanol. After washing, a peptide diluted with a 50%o-benzotriazol-1-yl-N,N,N,N-tetramethyluronium-hexafluoro-phosphate(HBTU)/DCM was added and a coupling reaction was conducted at roomtemperature for around 60 to 120 minutes. The above-described steps wererepeated for coupling of all the amino acids. After the completion ofthe reaction, the N-terminal Fmoc group was removed using 50% HBTU/DCM,followed by recovery of the free peptide from the resin by using 95%trifluoroacetic acid (TFA). The peptide so recovered was diluted with a5% acetic acid solution, followed by the purification by thereverse-phase HPLC.

Synthesis of Peptides (II), (III), (IV), (V) and (VI)

Peptides (II), (III), (IV), (V) and (VI) were synthesized by thesolid-phase method (tBoc) by using a peptide synthesizer ("Beckman990c").

An amino acid having an N-terminal protected with a t-butyloxycarbonyl(tBoc) group was coupled on a resin by the method which is describedbelow. Incidentally, a phenylacetamidomethyl (Pam) resin was used forthe synthesis of a peptide having a carboxyl group as a C-terminal and a4-methylbenzhydrylamine (MBHA) resin was used for the synthesis of apeptide having an amide group as a C-terminal.

First, the amino acid on the resin was deprotected at room temperaturefor 25 minutes by using 50% TFA/DCM, followed by washing twice withisopropanol, DIPEA and DCM, respectively. Then, a peptide diluted with50% DMF/DCM was added and coupling reaction was carried out at roomtemperature for around 60 to 120 minutes. The above-described steps wererepeated and coupling reactions of all the amino acids were completed.Peptide (IV) was obtained by recovering the free peptide from the resinby the HF method (J. M. Stewart et al., Pierce Chemical Co., Rockford,Ill., 1984), diluting the resulting free peptide with a 5% acetic acidsolution and then purifying the reverse-phase HPLC method. Peptide (II)was obtained by removing the tBoc group at the N-terminal by using 50%TFA/DCM, recovering the free peptide from the resin by the HF method,diluting with a 5% acetic acid solution, and then purifying by thereverse-phase HPLC method. Peptides (III), (V) and (V) were eachobtained by adding an acetyl group (Ac) to the N-terminal by using 20%acetic anhydride, recovering the free peptide by the HF method, dilutingwith a 5% acetic acid solution and then purifying by the reverse-phaseHPLC method.

Detection of the peptide purity thus synthesized

The purity of each peptide thus synthesized was determined using a C-18column (Vydac Corp.) by the reverse-phase HPLC method. With a lineargradient from 5% to 25% of acetonitrile/purified water as a mobilephase, the elution peak was detected by measuring an absorbance at 215nm.

FIG. 1 is a chart of the chromatogram of Peptide (I). Peptide (I)synthesized above had a purity of 100%.

FIG. 2 is a chart of the chromatogram of Peptide (II). Peptide (II)synthesized above had a purity of 99.6%.

FIG. 3 is a chart of the chromatogram of Peptide (III). Peptide (III)synthesized had a purity of 98.8%.

FIG. 4 is a chart of the chromatogram of Peptide (IV). Peptide (IV)synthesized above had a purity of 97.2%.

FIG. 5 is a chart of the chromatogram of Peptide (V). Peptide (V)synthesized above had a purity of 98.5%.

FIG. 6 is a chart of the chromatogram of Peptide (VI). Peptide (VI)synthesized had a purity of 98.4%.

Example 2 Measurement of the Suppressive Effect of Peptide (I) onAnti-OVA Antibody Production in Mice.

Day 5 and day 3 prior to the antigen administration, Peptide (I)composed of 9 amino acid residues as indicated by Sequence ID No. 1 inthe Sequence Listing of the present invention and a synthetic peptide 15composed of 15 amino acid residues derived from clone 17.2 TcR, whichpeptide had been reported by Mohapatra et al. as described above, 100 geach, were dissolved in 200 of physiological saline and each solutionthus obtained was subcutaneously administered to 9-week-old Balb/c mice.As a control, a non-administered group to which the same amount ofphysiological saline was administered was employed. In 200 l ofphysiological saline, 100 g of OVA (Sigma Chemical Co.) were dissolved,followed by subcutaneous administration. Blood was collected on day 21after antigen administration and an antibody titer was measured by themethod described below.

OVA was diluted to 10 g/ml with phosphate buffered saline (PBS) and 50 lportions of the solution were poured in a 96-well microplate. Thecoating operation was conducted overnight at 40° C. The microplate wasthen washed three times with a 0.05% Tween®-20-containing PBS (T-PBS).200 l portions of 0.5% casein-containing tris buffered physiologicalsaline (casein TBS) were poured in each well and the microplate wasallowed to stand at room temperature for one hour. The microplate wasthen washed three times with T-PBS. After the serum of the bloodcollected from the mice was diluted to 1/200 with 0.5% casein TBS, 50 lportions of the diluted serum were poured in each well and the plate wasallowed to stand for one hour (primary reaction).

After the completion of the primary reaction, the microplate was washedthree times with T-PBS. Fifty l portions of anti-mouseIgG-peroxidase-labeled antibody (TAGO Co.) diluted to 1/4000 with 0.5%casein TBS were poured in each well, followed by reaction at roomtemperature for one hour (secondary reaction). After the completion ofthe secondary reaction, the plate was washed three times again. Fifty lportions of a substrate liquid/chromogen (chromogen substrate:Behringwerke AG) were poured in each well, followed by reaction at roomtemperature for 30 minutes. After the completion of the reaction, 0.5Ndiluted sulfuric acid was added to terminate the reaction and anabsorbance at 450 nm was measured by a calorimeter ("BEP-II", product ofBehringwerke AG/Germany) with an absorbance at 650 nm as a control. Theresults are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Suppressive effect of Peptide (I) of this invention on anti-OVA antibody       production in mouse                                                            Treated with       Anti OVA Ig-G antibody titer (unit)                      ______________________________________                                        Non-administered group                                                                           59 ± 40.0                                                 Peptide 15 derived from clone 17.2 13.5 ± 12.0                             Peptide (I)  8.8 ± 10.8                                                  ______________________________________                                    

From the above results, it has been found that compared with thenon-administered group, antibody production was significantly suppressedin the mice to which synthetic peptide 15 derived from clone 17.2 andPeptide (I) of this invention were administered, respectively.Incidentally, each group consisted of 8 mice.

Example 3 Measurement of the Suppressive Effect of Peptides (I) to (VI)on Anti-KLH Antibody Production in Mice

Day 5 and day 3 prior to the antigen administration, 100 μg of eachPeptide (I) to (VI) of the present invention composed of 9 amino acidresidues indicated by Sequence ID No. 1 in the Sequence Listing weredissolved in 200 μl of physiological saline. The resulting solution wassubcutaneously administered to 9-week-old Balb/c mice. As a control, anon-administered group to which the same amount of physiological salinewas administered was employed. In 200 μl of physiological saline, 25 μgof KLH were dissolved as an antigen and the resulting solution wassubcutaneously administered. The blood was collected on day 21 afterantigen administration and an antibody titer was measured by the methodwhich is described below.

KLH was diluted to 10 μg/ml with a phosphate buffered physiologicalsaline (PBS). Fifty μl portions of the resulting solution were pouredinto a 96-well microplate and the plate was allowed to stand at 4iCovernight. After the microplate was washed three times with 0.05%Tween®-20-containing PBS (T-PBS), 200 μl portions of 0.5% caseintris-buffered physiological saline (casein TBS) were poured into wells,respectively. The microplate was then allowed to stand at roomtemperature for one hour. The microplate was washed three times withT-PBS and 50 μl portions of the mouse serum diluted to 1/200 with caseinTBS were poured into the wells, followed by reaction for one hour(primary reaction).

The resulting microplate was washed three times with T-PBS, to which 50μl portions of an anti-mouse IgG-peroxidase-labeled antibody (TAGO Co.)diluted to 1/4000 with casein TBS were poured. The reaction was effectedat room temperature for one hour (secondary reaction). After thecompletion of the secondary reaction, the plate was washed three timesagain. Fifty μl portions of a substrate liquid/chromogen (chromogensubstrate: Behringwerke AG) were poured into wells, respectively,followed by reaction at room temperature for 30 minutes. After thecompletion of the reaction, 0.5N diluted sulfuric acid was added toterminate the reaction and an absorbance at 450 nm was measured by acalorimeter ("BEP-II ", product of Behringwerke AG/Germany) with theabsorbance at 650 nm as a control. The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Suppressive effect of Peptides of this invention on anti-KLH                    antibody production                                                             Treated with Anti-KLH IgG antibody titer (unit ± SD)                   ______________________________________                                        Peptide (I)  78.7 ± 28.3                                                     Peptide (II) 48.4 ± 13.1                                                   Peptide (III) 50.1 ± 25.8                                                  Peptide (IV) 48.5 ± 24.9                                                   Peptide (V) 37.7 ± 17.7                                                    Peptide (VI) 29.5 ± 19.5                                                   Physiological saline 133.8 ± 48.2                                        ______________________________________                                    

From the above results, it has been found that compared with thenon-administered group to which physiological saline had beenadministered, antibody production was significantly suppressed in themice groups to which Peptides (I) to (VI) had been administered,respectively. Incidentally, each group consisted of 8 mice.

It has been understood from the results of Examples 2 and 3, thepeptides of the present invention had antigen non-specificimmunosuppressive action.

Example 4 Onset Suppressive Effect of Peptide (I) on EAE Model Mice

As an antigen, guinea pig spinal chord homogenate was employed. Theguinea pig spinal chord homogenate was diluted with PBS to be 6.6 mg/ml,followed by the addition of the equivalent amount of Freunds completeadjuvant. The resulting mixture was formed into an emulsion by anultrasonic sonicator. The resulting emulsion was administered to twoareas under the lateral skin in a total amount of 300 μl. Day 5 and day3 prior to antigen administration, and 5 days a week for 4 weeks afterantigen administration, the solution of Peptide (I) dissolved in 100 μlphysiological saline was subcutaneously administered to a 14-week oldSJL mouse. Incidentally, in order to increase an onset ratio, 400 μg/100μl of pertussis toxin (PTX) were intravenously administered as anenhancer on the day of antigen administration and two days after antigenadministration. Observation of the onset was performed from day 14 today 28 after primary antigen administration. The onset was scored basedon the following standard criteria [Pettinelli, C. B., Fritz, D. E., andMcFarlin, D. E. J. Immunl., 129, No. 3, 1024-1028 (1982)]. In Table 3,values of average score+standard deviation (SD) in each administrationgroup on day 22, day 25 and day 28 after primary antigen administrationare shown. Incidentally, each group consisted of 8 mice and Steels twotail method was used for statistical analysis.

Score 0: No abnormality

Score 1: Slight paralysis of hind leg with tail weakness

Score 2: Moderate paralysis of hind leg with tail weakness

Score 3: Total paralysis of hind leg

Score 4: Slight paralysis of forward leg with total paralysis of hindleg

Score 5: Total paralysis of all leg

Score 6: Death from paralysis

The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Onset suppressive effect of Peptide (I) of this invention                       on EAE model mice (average score ± SD)                                             Day 22       Day 25   Day 28                                        ______________________________________                                        25 μg/body                                                                           0.3 ± 0.4 0.1 ± 0.3                                                                           0.0 ± 0.0                                    5 μg/body 0.4 ± 0.7 1.0 ± 1.1 0.7 ± 0.8                           1 μg/body 1.6 ± 1.8 0.9 ± 1.1 1.8 ± 1.9                           physiological 3.6 ± 2.5 4.2 ± 2.6 4.2 ± 2.6                          saline                                                                      ______________________________________                                    

It has been recognized that the onset of EAE was suppressed by theadministration of synthetic Peptide (I) of the present invention.Particularly, in 5 μg/body administered group, the onset was suppressedon day 25 and day 28 with a significant difference of 5% compared withthe physiological saline administered group. In the 25 μg/bodyadministered group, the onset was suppressed from day 22 with asignificant difference of 5% compared with the physiological salineadministration group. Moreover, from day 25, the onset was suppressedwith a significant difference of 1%.

It has been found from Example 4 that the peptide of the presentinvention is effective for the treatment of multiple sclerosis.

Example 5 Suppressive Effect of Peptide (I) on Rat Carrageenin-InducedPaw Edema Model

To physiological saline, λ-carrageenin ("PICNIN-A", Zushi Chemical Co.)was added at the final concentration of 1 w/v %, and it was heated in aboiling water bath to dissolve I-carrageenin completely. The resultingsolution was then allowed to stand at room temperature. Before theexperiment, the solution was dissolved completely by heating in aboiling water bath and was allowed to stand in a water bath of about60iC until administration. Immediately after 50 μl of Peptide (I) of thepresent invention or physiological saline (non-administered group) wereadministered subcutaneously to the left leg of Splague-Dawley rats (6weeks old, male), 50 μl of the 1% carrageenin solution were administeredto the same site. The paw volume was measured using plethysmometer(product of BM Instrument Co.) every one hour for 5 hours aftercarrageenin administration. Incidentally, each group consisted of 10rats and Dunnets two tail method was used for statistical analysis. Theresults are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Suppressive effect of Peptide (I) of this invention on edema (%)                        1 hour       2 hours 5 hours                                        ______________________________________                                        0.2 μg/site                                                                          2.3          3.9     4.8                                              10 μg/site 30.2 37.3 27.5                                                ______________________________________                                    

It has been recognized that the onset of edema one hour after theadministration of carrageenin tended to be suppressed by theadministration of Peptide (I) of the present invention. In the 10μg/site administered group, the onset of edema was suppressed two hourson and after the administration of carrageenin with a significantdifference of 5% and the tendency to suppress the edema onset wasobserved five hours after the administration of carrageenin. From theabove, it has been found that the peptide of the present invention hasanti-inflammatory action. Moreover, the present inventors conducted thefollowing experiment in order to find effective low-molecular weightpeptides.

Example 6 Synthesis of Peptides

Peptides (VII), (VIII), (IX), (X), (XI), (XII), (XIII), (XIV) and (XV)were synthesized and purified in a similar manner to Example 1 by usinga peptide synthesizer ("Model 430A", ABI Co.).

    ______________________________________                                        Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 2) (VII)                           Ala-Ala-Leu-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 3) (VIII)                        Ala-Lys-Leu-Ala-Phe-Gly-Lys-Gly (Seq. ID No. 4) (IX)                          Ala-Lys-Leu-Thr-Phe-Ala-Lys-Gly (Seq. ID No. 5) (X)                           Ala-Lys-Leu-Thr-Phe-Gly-Ala-Gly (Seq. ID No. 6) (XI)                          Ala-Lys-Leu-Thr-Phe-Gly-Lys-Ala (Seq. ID No. 7) (XII)                         Ala-Lys-Ala-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 8) (XIII)                        Ala-Lys-Leu-Thr-Ala-Gly-Lys-Gly (Seq. ID No. 9) (XIV)                         Lys-Leu-Thr-Phe-Gly-Lys-Gly (Seq. ID No. 10) (XV)                           ______________________________________                                    

Detection of the purity of peptides so synthesized was performed.

The purity of each peptide so synthesized was detected in a similarmanner to Example 1.

FIG. 7 is a chart of the chromatogram of Peptide (VII). Peptide (VII) sosynthesized had a purity of 97.9%.

FIG. 8 is a chart of the chromatogram of Peptide (VIII). Peptide (VIII)so synthesized had a purity of 98.1%.

FIG. 9 is a chart of the chromatogram of Peptide (IX). Peptide (IX) sosynthesized had a purity of 97.5%.

FIG. 10 is a chart of the chromatogram of Peptide (X). Peptide (X) sosynthesized had a purity of 98.2%.

FIG. 11 is a chart of the chromatogram of Peptide (XI). Peptide (XI) sosynthesized had a purity of 97.6%.

FIG. 12 is a chart of the chromatogram of Peptide (XII). Peptide (XII)so synthesized had a purity of 97.5%.

FIG. 13 is a chart of the chromatogram of Peptide (XIII). Peptide (XIII)so synthesized had a purity of 98.2%.

FIG. 14 is a chart of the chromatogram of Peptide (XIV). Peptide (XIV)so synthesized had a purity of 98.6%.

FIG. 15 is a chart of the chromatogram of Peptide (XV). Peptide (XV) sosynthesized had a purity of 100%.

Example 7 Measurement of the Suppressive Effect of Peptides (I), (VII)to (XV) on the Anti-KLH Antibody Production in Mice

In a similar manner to Example 3, suppressive effects of Peptides (I),(VII) to (XV) on anti-KLH antibody production in mice were studied. Theresults are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        Treated with   Anti-KLH antibody titer (unit ± SD)                         ______________________________________                                        Peptide (I)    39.5 ± 16.1                                                   Peptide (VII) 25.9 ± 15.4                                                  Peptide (VIII) 44.9 ± 18.1                                                 Peptide (IX) 33.9 ± 13.7                                                   Peptide (X) 26.5 ± 22.0                                                    Peptide (XI) 19.9 ± 15.9                                                   Peptide (XII) 49.5 ± 40.1                                                  Peptide (XIII) 68.9 ± 25.1                                                 Peptide (XIV) 59.9 ± 32.8                                                  Peptide (XV) 86.4 ± 27.8                                                   Physiological saline 115.7 ± 61.3                                          (non-administered group)                                                    ______________________________________                                    

Compared with the non-administered group and Peptide (I) administeredgroup, the antibody production was significantly suppressed in the miceto which Peptide (VII), (IX), (X) or (XI) was administered. The antibodyproduction was, on the other hand, not suppressed in the mice to whichPeptide (VIII), (XII), (XIII), (XIV) or (XV) was administered, comparedwith Peptide (I) administered group. Incidentally, each group consistedof 8 mice.

From the above results, it has been found that the minimum peptidepermitting the suppression of the antibody production is composed of 8amino acid residues as indicated by the Sequence ID No. 2 of theSequence Listing.

Example 8 Suppressive effect of Peptides (I), (VIl) to (XIV) on theanti-OVA antibody production in mice; and identification of the minimumunit of peptide and essential amino acid exhibiting these effects.

In a similar manner to Example 2, the suppressive effects of Peptides(I), (VII) to (XIV) on the anti-OVA antibody production in mice weremeasured. The results are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Treated with    Anti-OVA antibody titer                                       ______________________________________                                        Peptide (I)     23.8 ± 12.4                                                  Peptide (VII) 20.9 ± 21.4                                                  Peptide (VIII) 65.3 ± 34.7                                                 Peptide (IX) 10.4 ± 6.3                                                    Peptide (X) 49.8 ± 28.4                                                    Peptide (XI) 15.3 ± 14.3                                                   Peptide (XII) 50.9 ± 26.7                                                  Peptide (XIII) 193.5 ± 155.9                                               Peptide (XIV) 90.9 ± 89.5                                                  Physiological saline 151.0 ± 25.3                                          (non-administered group)                                                    ______________________________________                                    

The antibody production of the mice to which Peptide (VII), (IX) or (XI)had been administered was significantly suppressed compared with thenon-administered group and Peptide (I) administered group. The antibodyproduction of the mice to which Peptide (XIII) or (XIV) has beenadministered was, on the other hand, not suppressed compared withPeptide (I) administered group. Incidentally, each group consisted of 8mice.

From Examples 7 and 8, it has been found that a peptide havingsuppressive effects on antigen nonspecific antibody production isrequired to have Ala, Leu and Phe at the first, third and fifthpositions of its amino acid sequence, respectively, for the antibodyproduction suppression.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 10                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -      Ala Lys Leu Thr Phe Gly Lys Gly - # Thr                                  1             - #  5                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -      Ala Lys Leu Thr Phe Gly Lys Gly                                          1             - #  5                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -      Ala Ala Leu Thr Phe Gly Lys Gly                                          1             - #  5                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - -      Ala Lys Leu Ala Phe Gly Lys Gly                                          1             - #  5                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - -      Ala Lys Leu Thr Phe Ala Lys Gly                                          1             - #  5                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - -      Ala Lys Leu Thr Phe Gly Ala Gly                                          1             - #  5                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - -      Ala Lys Leu Thr Phe Gly Lys Ala                                          1             - #  5                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - -      Ala Lys Ala Thr Phe Gly Lys Gly                                          1             - #  5                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - -      Ala Lys Leu Thr Ala Gly Lys Gly                                          1             - #  5                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - -      Lys Leu Thr Phe Gly Lys Gly                                              1             - #  5                                                   __________________________________________________________________________

We claim:
 1. A peptide having the following amino acidsequence:Ala-Xaa1-Leu-Xaa2-Phe-Xaa3-Xaa4-Xaa5-(Xaa6)n wherein Xaa1 andXaa4 each independently represents a naturally occurring amino acidresidue which has an alkyl or heteroalkyl side chain which may besubstituted by a hydroxy, amino or guanidyl group; Xaa2 and Xaa6 eachindependently represents a naturally occurring amino acid residue whichhas an alkyl or heteroalkyl side chain which may be substituted by ahydroxyl group; Xaa3 and Xaa5 each independently represents a naturallyoccurring amino acid residue which has a hydrophobic side chain; and nstands for 1 or 0, with the proviso that the case where Xaa2 representsglycine is excluded,or a derivative thereof.
 2. A peptide or derivativesthereof according to claim 1, wherein n stands for 1 or 0;Xaa1 and Xaa4each independently represents any one of Lys, Arg, His and Ala; Xaa2 andXaa6 each independently represents Thr or Ala; Xaa3 and Xaa5 eachindependently represents Gly or Ala.
 3. A peptide or derivatives thereofaccording to claim 1, which has an amino acid sequence selected from thegroup consisting of the following amino acidsequences:Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr,Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly, Ala-Ala-Leu-Thr-Phe-Gly-Lys-Gly,Ala-Lys-Leu-Ala-Phe-Gly-Lys-Gly, Ala-Lys-Leu-Thr-Phe-Ala-Lys-Gly,Ala-Lys-Leu-Thr-Phe-Gly-Ala-Gly and Ala-Lys-Leu-Thr-Phe-Gly-Lys-Ala. 4.A peptide or derivatives thereof according claim 1, wherein the aminoacid sequence is partially or entirely in the D-form.
 5. A peptide orderivatives thereof according claim 1, wherein the amino group at theN-terminal has been substituted with a protective group.
 6. A peptide orderivatives thereof according to claim 5, wherein the protective groupof the amino group at the N-terminal is an acetyl or t-butoxycarbonylgroup.
 7. A peptide or derivatives thereof according claim 1, whereinthe carboxyl group at the C-terminal is a carboxy derivative.
 8. Apeptide or derivatives thereof according to claim 7, wherein the carboxyderivative at the C-terminal is an amide group.
 9. A peptide orderivatives thereof according claim 1, which is a peptide selected fromthe group consisting of peptide represented by the following formulas(I) to (XII):

    Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr                        (I)

    DAla-DLys-DLeu-DThr-DPhe-Gly-DLys-Gly-DThr                 (II)

    Ac-Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr                     (III)

    tBoc-Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly-Thr                   (IV)

    Ac-DAla-DLys-DLeu-DThr-DPhe-Gly-DLys-Gly-DThr              (V)

    Ac-DAla-DLys-DLeu-DThr-DPhe-Gly-DLys-Gly-DThr-NH2          (VI)

    Ala-Lys-Leu-Thr-Phe-Gly-Lys-Gly                            (VII)

    Ala-Ala-Leu-Thr-Phe-Gly-Lys-Gly                            (VIII)

    Ala-Lys-Leu-Ala-Phe-Gly-Lys-Gly                            (IX)

    Ala-Lys-Leu-Thr-Phe-Ala-Lys-Gly                            (X)

    Ala-Lys-Leu-Thr-Phe-Gly-Ala-Gly                            (XI) and

    Ala-Lys-Leu-Thr-Phe-Gly-Lys-Ala                            (XII)

(wherein D represents a D-form, Ac represents an acetyl group and tBocrepresents a t-butoxycarbonyl group), or a derivative thereof.
 10. Acomposition comprising at least one peptide or derivatives thereof asclaimed in claim 1.